Bis-h-phosphinic acid derivatives as precursors to therapeutic bisphosphonates and uses thereof

ABSTRACT

A novel class of compounds, i.e., 1,1-bis-H-phosphinates (or 1,1-bis-H-phosphinic acid derivatives) are provided. Also provided are novel methods for producing 1,1-bis-H-phosphinates and 1,1-bis-H-phosphinate conjugates. These compounds and conjugates are used as precursors of bisphosphonates and bisphosphonate conjugates, respectively, or as prodrugs directly for treating bone-related and various other diseases.

BACKGROUND OF THE INVENTION

[0001] 1. Funding Legend:

[0002] This invention was produced in part using funds through grant No. P-1435 from the Robert A. Welch Foundation.

[0003] 2. Field of the Invention:

[0004] The present invention relates generally to the field of chemistry and drug therapy. More particularly, the present invention provides novel bis-H-phosphinic acid derivatives and methods of producing bis-H-phosphinic acid derivatives. The present invention also provides methods of producing drug or protein conjugates using bis-H-phosphinic acid derivatives and methods of treating bone-related and various other diseases using such derivatives or conjugates.

[0005] 3. Description of Related Art:

[0006] Bisphosphonates are used to target molecules to the bone. Pharmaceutical compounds derived from bisphosphonates are used to treat bone cancer, bone infection or deliver steroids to the bone to treat osteoporosis. Bisphosphonates are not only important drugs for the treatment of bone-related diseases, but also important drugs for various other diseases such as high cholesterol. They are currently $1 billion market worldwide. Although they are generally 1-hydroxy-substituted, these compounds have a major limitation. That is, they display poor oral bioavailability because most of the compounds are excreted. Also high dosage causes serious side effects.

[0007] Several pharmaceutical companies have been trying to modify bisphosphonates to improve their biological activities. The approach commonly used is to mask the charge of the phosphonate to increase penetration through membranes. However, extensive manipulation is required to modify bisphosphonates which have 3-4 charges at physiological pH.

[0008] Bisphosphinates (or phosphinic acid derivatives) are precursors to bisphosphonates. Presently, bisphosphinates oxidize into corresponding bisphosphonates by cumbersome multistep synthesis. Bisphosphinates have been prepared under radial conditions. For example, Nifant'ev (J. Gen. Chem. USSR 1986, 56, 680) taught reacting sodium hypophosphite or hypophosphorous acid with alkynes in the presence of peroxides and extensive heat to prepare biophosphinates. Low yields of 1,2-bis-H-phosphinates were sometime obtained from Nifant'ev's method.

[0009] Therefore, there is an increasing need for a simple, expeditious and effective method for synthesizing bisphosphonates with increased bioavailability.

SUMMARY OF THE INVENTION

[0010] With the foregoing in mind, the present invention provides a novel class of compounds, i.e., 1,1-bis-H-phosphinates (or 1,1-bis-H-phosphinic acid derivatives). Also, the present invention provides a novel method for producing 1,1-bis-H-phosphinates. These compounds are used as precursors of bisphosphonates through chemical or in vivo biological oxidation.

[0011] In one embodiment of the present invention, there is provided a bis-H-phosphinate compound having a structure of

[0012] In the structure, R includes Na, H, RhNH₃, OH and AlK, and R′ includes COOH, NH₂, epoxide, OH, CHO, SH, CH₂NH₂, CH₂COO-steroid, CH₂-farnesyl, CH₂-geranyl, N-fluoroquinolones, CH₂S-albumin and NMe₂CF₃COOH. Preferably, this compound is a 1,1-bis-H-phosphinate.

[0013] In another embodiment of the present invention, there is provided a method for producing a bis-H-phosphinate compound. In this method, a hypophosphorous derivative reacts with an alkyne in the presence of a radical initiator at ambient temperature and pressure conditions. The reaction can be carried out, for example, at room temperature in an open container. Preferably, the radical initiator is an organoborane catalyst, for example, triethylborane.

[0014] In still another embodiment of the present invention, there is provided a method for producing a bis-H-phosphinate conjugate. In this method, a hypophosphorous derivative first reacts with an alkyne in the presence of a radical initiator at room temperature in an open container to produce a bis-H-phosphinate compound. The bis-H-phosphinate then reacts with a compound having a pendant functional group to produce a bis-H-phosphinate conjugate. Preferably, the compound having a pendant functional group is a pharmaceutical compound or a protein. An example of the functional group is an alkyne group.

[0015] In yet another embodiment of the present invention, there is provided a method for treating an individual having a bone-related disease by administering to the individual an effective dose of a bis-H-phosphinate compound or a bis-H-phosphinate conjugate. Same compound or conjugate can also be used for treating an individual having high cholesterol.

BRIEF DESCRIPTION OF THE DRAWING

[0016] Some of the features, advantages, and benefits of the present invention having been stated, others will become apparent as the description proceeds when taken in conjunction with the accompanying drawings in which:

[0017]FIG. 1 is a scheme illustrating the process of modifying bisphophonates using bisphosphinates as precursors;

[0018]FIG. 2 is a scheme illustrating the process of preparing 1,1-bis-H-phosphinate conjugates;

[0019]FIG. 3 is a scheme illustrating the process of first preparing disodium 1,1-bis-H-phosphinates and further preparing 1,1-bisphophonates;

[0020]FIG. 4 is a list of bisphosphonates that display important biological activities;

[0021]FIG. 5 is a scheme illustrating the process of preparing 1,2-bis-H-phosphinates; and

[0022]FIG. 6 is a scheme illustrating the process of preparing a disodium 1,1-bis-H-phosphinate from steroid conjugate.

DETAILED DESCRIPTION

[0023] The present invention will now be described more fully hereinafter with reference to the accompanying drawings which illustrated preferred embodiments of the invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the illustrated embodiments set forth herein. Rather, these illustrated embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0024] A new class of compounds is prepared in one step from alkynes and sodium hypophosphite (or other hypophosphorous derivatives), under the influence of an organoborane catalyst, at room temperature (RT), and in an open flask (air) (reaction (1)).

[0025] With R=Na, 1,1-bis-H-phosphinate (compounds 1, the major product) precipitates spontaneously and further can be isolated in pure form by simple filtration. Meanwhile, 1,2-bis-H-phosphinate (compounds 2) is also produced. Other hypophosphorous derivatives (for example, R=H, PhNH₃, or Alk) undergo addition to alkynes under similar conditions, although 1,2-bis-H-phosphinate is usually the major product.

[0026] Both 1,1-bis-H-phosphinates (compounds 1) and reaction (1) have not been described previously. 1,1-bis-H-phosphinates are precursors to bisphosphonates (compounds 3) (reaction (2)). This reaction occurs under either chemical or metabolic oxidation.

[0027] 1,1-bis-H-phosphinates with a reaction group (compounds 4) can be converted into 1,1-bisphosphonates (compounds 5), which have been used previously to modify drugs or proteins to construct drug or protein 1,1-bis-H-phosphonate conjugates (compounds 6) (FIG. 1). Examples of the reactive group (RG) include COOH, NH₂, and SH.

[0028] 1,1-bis-H-phosphinate conjugates are prepared from compounds suitably functionalized with a pendant alkyne group (FIG. 2). These conjugates can be easily transformed into bisphosphonates conjugates which are previously difficult to obtain. 1,1-Bis-H-phosphinate conjugates can also be used directly as prodrugs of the corresponding bisphosphonates. Examples of functional group (FG) include OH, NH₂, SH, and COOH, while examples of reactive group (RG) include COOH, NH₂, epoxide, OH, and CHO.

[0029] Using sodium hypophosphite hydrate (NaH₂PO₂.H₂O) as a substrate, disodium 1,1-bis-H-phosphinate is prepared when an alkyne is added to a solution of sodium hypophosphite hydrate at room temperature in an open flask. 1,1-bis-H-phosphinate is then transformed to 1,1-bis-H-phosphonate under chemical or in vivo biological oxidation (FIG. 3). Examples of alkynes are listed in both FIG. 3 and Table 1. TABLE 1 Alkynes Used in Formation of Disodium 1,1-Bis-H-phosphinates Alkynes Yield % δ ³¹P NMR in D₂O

48 22.5

39 28.7

48 26.5

35 27.7

37 28.1

39

44 29.2

50 26.1

50 26.3

21 26.0

52 24.6

[0030] Some bisphosphonates display important biological activities (e.g. against bone disease, osteoporosis, or as cholesterol lowering reagents) (FIG. 4). Currently available syntheses of these bisphosphonates are lengthy and difficult. The task of masking the initial charge of the phosphonates is enormous. In contrast, the present invention provides a simple process to synthesize the bisphosphonates in one or two steps from readily available alkynes. Also, the 1,1-bis-H-phosphinates of the present invention are only dianionic at physiological pH and therefore much more lipophilic. There are suggestions in the literature that H-phosphinic acids undergo biological (metabolic) oxidation to become the actual bisphosphonates (Froestl, et al., J Med. Chem. 1995, 38, 3297). Because they have reduced polarity compared to the corresponding bisphosphonates, 1,1-bis-H-phosphinates should possess increased bioavailability, including oral delivery. Therefore the 1,1-bis-H-phosphinates of the present invention are ideal prodrugs and might be used directly for therapeutic applications.

[0031] The present invention provides a bis-H-phosphinate compound having a structure of

[0032] In the structure, R includes Na, H, RhNH₃, OH and AlK, and R′ includes COOH, NH₂, epoxide, OH, CHO, SH, CH₂NH₂, CH₂COO-steroid, CH₂-farnesyl, CH₂-geranyl, N-fluoroquinolones, CH₂S-albumin and NMe₂CF₃COOH. Preferably, this compound is a 1,1-bis-H-phosphinate.

[0033] The present invention also provides a method for producing a bis-H-phosphinate compound. In this method, a hypophosphorous derivative reacts with an alkyne in the presence of a radical initiator at room temperature in an open container. Further preferably, this method includes filtering the reaction mixture to obtain the bis-H-phosphinate compound in the filtrate. Still preferably, the radical initiator is an organoborane catalyst, for example, triethylborane.

[0034] The present invention further provides a method for producing a bis-H-phosphinate conjugate. In this method, a hypophosphorous derivative first reacts with an alkyne in the presence of a radical initiator at room temperature in an open container to produce a bis-H-phosphinate compound. The bis-H-phosphinate then reacts with a compound having a pendant functional group to produce a bis-H-phosphinate conjugate. Preferably, the compound having a pendant functional group is a pharmaceutical compound or a protein. A representative example of the functional group is an alkyne group.

[0035] Furthermore, the present invention provides a method for treating an individual having a bone-related disease by administering to the individual an effective dose of a bis-H-phosphinate compound or a bis-H-phosphinate conjugate. Same compound or conjugate can also be used for treating an individual having high cholesterol.

[0036] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.

EXAMPLE 1

[0037] Preparation of Disodium 1,1-Bis-H-phosphinates in Methanol

[0038] An alkyne (2 mmol) was added to a solution of NaH₂PO₂H₂.O (5 mmol) in reagent grade methanol (10 mL). Triethylborane (1 M in hexane, 2 mmol) was then added in one portion to the stirred solution at room temperature in open container (i.e., with air). The reaction mixture rapidly became cloudy, and a white precipitate formed. After 2 hours, the mixture was filtered and the solid was washed with cold methanol or ethanol. NMR analysis of the solid at this stage generally showed at least 90% purity. If the solid was sufficiently pure (ca. 98%), it was then dried in vacuum for 24 h over P₂O₅. The disodium 1,1-bis-H-phosphinates are generally highly hygroscopic and obtained as white amorphous powders.

[0039] The filtrate also contained unreacted NaH₂PO₂, and variable amounts of 1,2-bis-H-phosphinates. For certain substrates, small amounts of monoaddition products (alkenyl-H-phosphinates) may also be observed in the filtrate.

EXAMPLE 2

[0040] Preparation of Disodium 1,1-Bis-H-phosphinates in Methanol/Acetone

[0041] The reaction was conducted exactly the same as in Example 1, except that methanol (10 mL) was replaced with a solution of methanol (8 mL) and acetone (2 mL). Isolated yields of the disodium 1,1-bis-H-phosphinate were improved without affecting purity.

EXAMPLE 3

[0042] Preparation of Disodium 1,1-Bis-H-Phosphinates from Amine-Containing Alkynes

[0043] With the exception of propargyl amine, which reacted well with the procedures in Examples 1 & 2, more basic amines such as 1-dimethylamino-2-propyne were poor substrates. However, addition of CF₃COOH (1.0-1.2 eq. per basic nitrogen) to the reaction mixture prior to addition of Et₃B allowed the reaction to occur in good yield. It is hypothesized that the autocatalytic oxidation of the borane was slowed by complexing to the amine in the absence of acid, and that the efficiency of the radical reaction dropped. The 1,1-bis-H-phosphinate was also obtained as a white or tan precipitate.

EXAMPLE 4

[0044] Formation of the 1,2-Bis-H-phosphinates

[0045] When anilinium hypophosphite (PhNH₃O₂PH₂) was used in place of sodium hypophosphite, no precipitates formed, and the major product was the 1,2-isomer (FIG. 5). With 1-decyne, 1,2-isomer was 50% yield, whereas 1,1-isomer was 15% yield. The product was isolated after esterification of the crude mixture to the bis-alkyl-1,2-bis-H-phosphinate and chromatographic purification.

[0046] In detail, an alkyne (2 mmol) was added to a solution of PhNH₃O₂PH₂ (5 mmol) in reagent grade methanol (10 mL). Triethylborane (1 M in hexane, 2 mmol) was then added in one portion to the stirred solution at room temperature in open container. The reaction mixture was stirred for 2 hours, then concentrated under reduced pressure to brown oil. Tetraethyl orthosilicate (5 mmol) and toluene (10 mL) were then added. The mixture was refluxed under N₂ for 18-24 h. Concentration in vacuum and chromatographic purification afforded the diethyl- 1,2-bis-H-phosphinate.

EXAMPLE 5

[0047] Preparation of a Disodium 1,1-Bis-H-phosphinate from Steroid Conjugate

[0048] Conjugation of Estrone: Propargyl chlorofornate (0.80 mL, 8.2 mmol), was added dropwise to a solution of estrone (2.115 g, 7.8 mmol) and anhydrous pyridine (2.0 mL, 24.7 mmol) in dry CH₂Cl₂ (20 mL) at room temperature, under N₂. The mixture was stirred for 12 h, then EtOAc and aqueous HCl (0.5 N) were added. The organic layer was washed with aqueous NaHCO₃ (2 X) and brine (1 X). Drying over MgSO₄ and concentration afforded the estrone conjugate as a slightly impure white solid (2.566 g). This was used without purification in the radical reaction.

[0049] Radical Reaction: The steroid conjugate (2.566 g, 7.28 mmol) was dissolved in a mixture of EtOAc (20 mL), acetone (20 mL), and MeOH (30 mL). Since the steroid conjugate was not very soluble in MeOH, EtOAc was added. Sodium hypophosphite (2.304 g, 21.7 mmol) was added and the mixture was almost homogeneous. Et₃B (1 M in hexane, 8.0 mL, 8 mmol) was added in one portion, at room temperature and in air. The stirred mixture immediately became cloudy. After 2.5 h, the precipitate was filtered. The white powder contained the 1,1-isomer as the major product, but some 1,2-isomer was also present, as well as a small amount of sodium hypophosphite. The powder was triturated with MeOH (15 mL) for 2 h. Filtration gave the 1,1-isomer product as a white powder (1.990 g, 52%) in good purity after drying in vacuum over P₂O₅.

[0050] The process of conjugation of estrone and further radical reaction of the steroid conjugate is shown in FIG. 6.

EXAMPLE 6

[0051] Preparation of 1,1-Bis-H-phosphinic Acids from the Sodium Salt

[0052] The bis-sodium-bis-H-phosphinate salt can be converted to the corresponding acid by acidification with a mineral acid. The most convenient method to avoid separation of inorganic salts involved the use of a polymeric acid such as Amberlite H⁺. The sodium salt was suspended in methanol, and Amberlite H⁺ was added. The suspension was stirred vigorously at room temperature, and the resin was filtered. Concentration of the filtrate afforded the corresponding 1,1-bis-H-phosphinic acids as clear, slightly yellow oil, in quantitative yield.

EXAMPLE 7

[0053] Esterification of Disodium 1,1-Bis-H-phosphinates

[0054] To a suspension of the salt in dry CH₃CN was added anhydrous pyridine, EtOH, and pivaloyl chloride, at room temperature and under nitrogen. An exothermic reaction immediately took place. After 4 h, the cloudy mixture was concentrated to a white solid, and partitioned between EtOAc and aqueous HCl (1 N). The organic layer was washed successively with saturated aqueous NaHCO₃ and brine. Drying over MgSO₄ and concentration afforded the ester as colorless oil.

EXAMPLE 8

[0055] Oxidation of the 1,1-Bis-H-phosphinates into the Corresponding Bisphosphonates

[0056] Because solutions of the disodium 1,1-bis-H-phosphinates were neutral, these compounds were surprisingly resistant to oxidation with bleach or 30% aqueous hydrogen peroxide at room temperature. An aqueous solution of bisphosphinate treated with these oxidants showed no detectable oxidation after 24 h at room temperature. However, gentle heating of the solution at 50-80° C. resulted in quantitative oxidation to the corresponding bisphosphonate. On the other hand, the 1,1-bis-H-phosphinic acids were readily oxidized at room temperature.

[0057] In the drawings and specification, there have been disclosed a typical preferred embodiment of the invention, and although specific terms are employed, the terms are used in a descriptive sense only and not for purposes of limitation. The invention has been described in considerable detail with specific reference to these illustrated embodiments. It will be apparent, however, that various modifications and changes can be made within the spirit and scope of the invention as described in the foregoing specification and as defined in the appended claims. 

What is claimed is:
 1. A bis-H-phosphinate compound having a structure

wherein R is selected from the group consisting of Na, H, RhNH₃, OH and AlK, and R′ is selected from the group consisting of COOH, NH₂, epoxide, OH, CHO, SH, CH₂NH₂, CH₂COO-steroid, CH₂-farnesyl, CH₂-geranyl, N-fluoroquinolones, CH₂S-albumin and NMe₂CF₃COOH.
 2. The bis-H-phosphinate compound of claim 1, wherein the compound is a 1,1-bis-H-phosphinate.
 3. A method for producing a bis-H-phosphinate compound, the method comprising the step of: reacting a hypophosphorous derivative with an alkyne in the presence of a radical initiator at room temperature, thereby producing a bis-H-phosphinate compound.
 4. The method of claim 3, wherein the reaction occurs in an open container.
 5. The method of claim 3, wherein the radical initiator is an organoborane catalyst.
 6. The method of claim 5, wherein the organoborane catalyst is a trialkylborane.
 7. The method of claim 6, wherein the trialkylborane is triethylborane.
 8. The method of claim 3, the method further comprising the step of: filtering reaction mixture upon completion of the reaction, wherein the filtrate contains the bis-H-phosphinate compound.
 9. A method for producing a bis-H-phosphinate conjugate, the method comprising the steps of: reacting a hypophosphorous derivative with an alkyne in the presence of a radical initiator at room temperature, thereby producing a bis-H-phosphinate compound; reacting the bis-H-phosphinate compound with a compound having a pendant functional group, thereby producing a bis-H-phosphinate conjugate.
 10. The method of claim 9, wherein the hypophosphorous derivative reacts with the alkyne in an open container.
 11. The method of claim 9, wherein the radical initiator is an organoborane catalyst.
 12. The method of claim 11, wherein the organoborane catalyst is a trialkylborane.
 13. The method of claim 12, wherein the trialkylborane is triethyl borane.
 14. The method of claim 9, wherein the pendant functional group is an alkyne group.
 15. The method of claim 9, wherein the compound having a pendant functional group is a pharmaceutical compound.
 16. The method of claim 9, wherein the compound having a pendant functional group is a protein.
 17. A method for treating an individual having a bone-related disease, the method comprising the step of: administering to the individual an effective dose of a bis-H-phosphinate compound or a bis-H-phosphinate conjugate.
 18. The method of claim 17, wherein the bis-H-phosphinate compound is a 1,1-bis-H-phosphinate having a structure

wherein R includes Na, H, RhNH₃, OH and AlK, and R′ includes COOH, NH₂, epoxide, OH, CHO, SH, CH₂NH₂, CH₂COO-steroid, N-fluoroquinolones, CH₂S-albumin and NMe₂CF₃COOH.
 19. The method of claim 17, wherein the bis-H-phosphinate conjugate is a conjugate of a bis-H-phosphinate compound with a compound having a pendant functional group.
 20. The method of claim 19, wherein the pendant functional group is an alkyne group.
 21. The method of claim 19, wherein the compound having a pendant functional group is a pharmaceutical compound.
 22. The method of claim 19, wherein the compound having a pendant functional group is a protein.
 23. The method of claim 17, wherein the bone-related disease includes osteoporosis, bone resorption, bone cancer, and bone infection.
 24. A method for treating an individual having high cholesterol, the method comprising the step of: administering to the individual an effective dose of a bis-H-phosphinate compound or a bis-H-phosphinate conjuagte.
 25. The method of claim 24, wherein the bis-H-phosphinate compound is a 1,1-bis-H-phosphinate having a structure

wherein R is selected from the group consisting of Na, H, RhNH₃, OH and AlK, and R′ is selected from the group consisting of CH₂-farnesyl and CH₂-geranyl.
 26. The method of claim 24, wherein the bis-H-phosphinate conjugate is a conjugate of a bis-H-phosphinate compound with a compound having a pendant functional group.
 27. The method of claim 26, wherein the pendant functional group is an alkyne group.
 28. The method of claim 26, wherein the compound having a pendant functional group is a pharmaceutical compound.
 29. The method of claim 26, wherein the compound having a pendant functional group is a protein.
 30. A method for treating an individual for therapeutic purposes, the method comprising the step of: administering to the individual an effective dose of a bis-H-phosphinate compound or a bis-H-phosphinate conjugate; wherein the bis-H-phosphinate compound or conjugate is oxidized into a corresponding bisphosphonate compound in vivo through biological oxidation.
 31. The method of claim 30, wherein the bis-H-phosphinate compound is a 1,1-bis-H-phosphinate having a structure

wherein R is selected from the group consisting of Na, H, RhNH₃, OH and AlK, and R′ is selected from the group consisting of CH₂-farnesyl and CH₂-geranyl.
 32. The method of claim 30, wherein the bis-H-phosphinate conjugate is a conjugate of a bis-H-phosphinate compound with a compound having a pendant functional group.
 33. The method of claim 32, wherein the pendant functional group is an alkyne group.
 34. The method of claim 32, wherein the compound having a pendant functional group is a pharmaceutical compound.
 35. The method of claim 32, wherein the compound having a pendant functional group is a protein. 